Absent in Melanoma 2 (AIM2) is an important mediator of interferon-dependent and -independent HLA-DRA and HLA-DRB gene expression in colorectal cancers

Absent in Melanoma 2 (AIM2) is a member of the HIN-200 family of hematopoietic, IFN-inducible, nuclear proteins, associated with both, infection defense and tumor pathology. Recently, AIM2 was found to act as a DNA sensor in innate immunity. In addition, we and others have previously demonstrated a high frequency of AIM2-alterations in microsatellite unstable (MSI-H) tumors. To further elucidate AIM2 function in colorectal tumors, we here addressed AIM2-responsive target genes by microarray based gene expression profiling of 22 244 human genes. A total of 111 transcripts were significantly upregulated, whereas 80 transcripts turned out to be significantly downregulated in HCT116 cells, constitutively expressing AIM2, compared with AIM2-negative cells. Among the upregulated genes that were validated by quantitative PCR and western blotting we recognized several interferon-stimulated genes (ISGs: IFIT1, IFIT2, IFIT3, IFI6, IRF7, ISG15, HLA-DRA, HLA-DRB, TLR3 and CIITA), as well as genes involved in intercellular adhesion and matrix remodeling. Expression of ISGs correlated with expression of AIM2 in 10 different IFN-γ treated colorectal cancer cell lines. Moreover, small interfering RNA-mediated knock-down of AIM2 resulted in reduced expression of HLA-DRA, HLA-DRB and CIITA in IFN-γ-treated cells. IFN-γ independent induction of HLA-DR genes and their encoded proteins was also demonstrated upon doxycyclin-regulated transient induction of AIM2. Luciferase reporter assays revealed induction of the HLA-DR promoter upon AIM2 transfection in different cell lines. STAT-signaling was not involved in IFN-γ independent induction of ISGs, arguing against participation of cytokines released in an autostimulating manner. Our data indicate that AIM2 mediates both IFN-γ dependent and independent induction of several ISGs, including genes encoding the major histocompatibility complex (MHC) class II antigens HLA-DR-α and -β. This suggests a novel role of the IFN/AIM2/ISG cascade likewise in cancer cells

Nonsense Mediated Decay Resistant Mutations Are a Source of Expressed Mutant Proteins in Colon Cancer Cell Lines with Microsatellite Instability

BACKGROUND: Frameshift mutations in microsatellite instability high (MSI-High) colorectal cancers are a potential source of targetable neo-antigens. Many nonsense transcripts are subject to rapid degradation due to nonsense-mediated decay (NMD), but nonsense transcripts with a cMS in the last exon or near the last exon-exon junction have intrinsic resistance to nonsense-mediated decay (NMD). NMD-resistant transcripts are therefore a likely source of expressed mutant proteins in MSI-High tumours. METHODS: Using antibodies to the conserved N-termini of predicted mutant proteins, we analysed MSI-High colorectal cancer cell lines for examples of naturally expressed mutant proteins arising from frameshift mutations in coding microsatellites (cMS) by immunoprecipitation and Western Blot experiments. Detected mutant protein bands from NMD-resistant transcripts were further validated by gene-specific short-interfering RNA (siRNA) knockdown. A genome-wide search was performed to identify cMS-containing genes likely to generate NMD-resistant transcripts that could encode for antigenic expressed mutant proteins in MSI-High colon cancers. These genes were screened for cMS mutations in the MSI-High colon cancer cell lines. RESULTS: Mutant protein bands of expected molecular weight were detected in mutated MSI-High cell lines for NMD-resistant transcripts (CREBBP, EP300, TTK), but not NMD-sensitive transcripts (BAX, CASP5, MSH3). Expression of the mutant CREBBP and EP300 proteins was confirmed by siRNA knockdown. Five cMS-bearing genes identified from the genome-wide search and without existing mutation data (SFRS12IP1, MED8, ASXL1, FBXL3 and RGS12) were found to be mutated in at least 5 of 11 (45%) of the MSI-High cell lines tested. CONCLUSION: NMD-resistant transcripts can give rise to expressed mutant proteins in MSI-High colon cancer cells. If commonly expressed in primary MSI-High colon cancers, MSI-derived mutant proteins could be useful as cancer specific immunological targets in a vaccine targeting MSI-High colonic tumours

Maternal psychological distress in primary care and association with child behavioural outcomes at age three

Observational studies indicate children whose mothers have poor mental health are at increased risk of socio-emotional behavioural difficulties, but it is unknown whether these outcomes vary by the mothers’ mental health recognition and treatment status. To examine this question, we analysed linked longitudinal primary care and research data from 1078 women enrolled in the Born in Bradford cohort. A latent class analysis of treatment status and self-reported distress broadly categorised women as (a) not having a common mental disorder (CMD) that persisted through pregnancy and the first 2 years after delivery (N = 756, 70.1 %), (b) treated for CMD (N = 67, 6.2 %), or (c) untreated (N = 255, 23.7 %). Compared to children of mothers without CMD, 3-year-old children with mothers classified as having untreated CMD had higher standardised factor scores on the Strengths and Difficulties Questionnaire (d = 0.32), as did children with mothers classified as having treated CMD (d = 0.27). Results were only slightly attenuated in adjusted analyses. Children of mothers with CMD may be at risk for socio-emotional and behavioural difficulties. The development of effective treatments for CMD needs to be balanced by greater attempts to identify and treat women. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00787-015-0777-2) contains supplementary material, which is available to authorized users

Screening for Microsatellite Instability Identifies Frequent 3′-Untranslated Region Mutation of the RB1-Inducible Coiled-Coil 1 Gene in Colon Tumors

BACKGROUND: Coding region microsatellite instability (MSI) results in loss of gene products and promotion of microsatellite-unstable (MSI-H) carcinogenesis. Recent studies have indicated that MSI within 3'-untranslated regions (3'UTRs) may post-transcriptionally dysregulate gene products. Within this context, we conducted a broad mutational survey of 42 short 3'UTR microsatellites (MSs) in 45 MSI-H colorectal tumors and their corresponding normal colonic mucosae. METHODOLOGY/PRINCIPAL FINDINGS: In order to estimate the overall susceptibility of MSs to MSI in MSI-H tumors, the observed MSI frequency of each MS was correlated with its length, interspecies sequence conservation level, and distance from some genetic elements (i.e., stop codon, polyA signal, and microRNA binding sites). All MSs were stable in normal colonic mucosae. The MSI frequency at each MS in MSI-H tumors was independent of sequence conservation level and distance from other genetic elements. In contrast, MS length correlated significantly with MSI frequency in MSI-H tumors (r=0.86, p=7.2x10(-13)). 3'UTR MSs demonstrated MSI frequencies in MSI-H tumors higher than the 99% upper limit predicted by MS length for RB1-inducible coiled-coil 1(RB1CC1, mutation frequency 68.4%), NUAK family SNF1-like kinase 1(NUAK1, 31.0%), and Rtf1, Paf1/RNA polymerase II complex component, homolog (RTF1, 25.0%). An in silico prediction of RNA structure alterations was conducted for these MSI events to gauge their likelihood of affecting post-transcriptional regulation. RB1CC1 mutant was predicted to lose a microRNA-accessible loop structure at a putative binding site for the tumor-suppressive microRNA, miR-138. In contrast, the predicted 3'UTR structural change was minimal for NUAK1- and RTF1 mutants. Notably, real-time quantitative RT-PCR analysis revealed significant RB1CC1 mRNA overexpression vs. normal colonic mucosae in MSI-H cancers manifesting RB1CC1 3'UTR MSI (9.0-fold; p = 3.6x10(-4)). CONCLUSIONS: This mutational survey of well-characterized short 3'UTR MSs confirms that MSI incidence in MSI-H colorectal tumors correlates with MS length, but not with sequence conservation level or distance from other genetic elements. This study also identifies RB1CC1 as a novel target of frequent mutation and aberrant upregulation in MSI-H colorectal tumors. The predicted loss of a microRNA-accessible structure in mutant RB1CC1 RNA fits the hypothesis that 3'UTR MSI involves in aberrant RB1CC1 posttranscriptional upregulation. Further direct assessments are indicated to investigate this possibility.Bogdan C. Paun, Yulan Cheng, Barbara A. Leggett, Joanne Young, Stephen J. Meltzer, Yuriko Mor

Mutations in TGFbeta-RII and BAX mediate tumor progression in the later stages of colorectal cancer with microsatellite instability

Abstract Background Microsatellite instability (MSI) occurs in 15% of colorectal cancers (CRC). The genetic targets for mutation in the MSI phenotype include somatic mutations in the transforming growth factor beta receptor typeII (TGFbetaRII), BAX, hMSH3 and hMSH6. It is not clear how mutations of these genes mediate tumor progression in the MSI pathway, and the temporal sequence of these mutations remains uncertain. In this study, early stage CRCs were examined for frameshift mutations in these target genes, and compared with late stage tumors and CRC cell lines. Methods We investigated 6 CRC cell lines and 71 sporadic CRCs, including 61 early stage cancers and 10 late stage cancers. Mutations of repetitive mononucleotide tracts in the coding regions of TGFbetaRII, BAX, hMSH3, hMSH6, IGFIIR and Fas antigen were identified by direct sequencing. Results Thirteen (18.3%) of 71 CRC, including 9/61 (14.7%) early stage cancers and 4/10 (40%) late stage cancers, were identified as MSI and analyzed for frameshift mutations. No mutation in the target genes was observed in any of the 9 early stage MSI CRCs. In contrast, frameshift mutations of TGFbetaRII, BAX, hMSH3 and hMSH6 were present in 3/4 late stage MSI tumors. There is a statistical association (p = 0.014) between mutation in any one gene and tumor stage. Conclusions TGFbetaRII, BAX, hMSH3 and hMSH6 mutations are relatively late events in the genesis of MSI CRCs. The frameshift mutations in these target genes might mediate progression from early to late stage cancer, rather than mediating the adenoma to carcinoma transition.</p

Marsh macrophyte responses to inundation anticipate impacts of sea-level rise and indicate ongoing drowning of North Carolina marshes

In situ persistence of coastal marsh habitat as sea level rises depends on whether macrophytes induce compensatory accretion of the marsh surface. Experimental planters in two North Carolina marshes served to expose two dominant macrophyte species to six different elevations spanning 0.75 m (inundation durations 0.4–99 %). Spartina alterniflora and Juncus roemerianus exhibited similar responses—with production in planters suggesting initial increases and then demonstrating subsequent steep declines with increasing inundation, conforming to a segment of the ecophysiological parabola. Projecting inundation levels experienced by macrophytes in the planters onto adjacent marsh platforms revealed that neither species occupied elevations associated with increasing production. Declining macrophyte production with rising seas reduces both bioaccumulation of roots below-ground and baffle-induced sedimentation above-ground. By occupying only descending portions of the parabola, macrophytes in central North Carolina marshes are responding to rising water levels by progressive declines in production, ultimately leading to marsh drowning

Mutations in the WTX - gene are found in some high-grade microsatellite instable (MSI-H) colorectal cancers

Background: Genetically, colorectal cancers (CRCs) can be subdivided into tumors with chromosomal instability (CIN) or microsatellite instability (MSI). In both types of CRCs genes that are involved in the degradation of beta-CATENIN are frequently mutated. Whereas in CIN CRCs APC (Adenomatous Polyposis Coli) is affected in most cases, high grade MSI (MSI-H) CRCs frequently display mutations in various genes, like the APC-, AXIN2- or CTNNBI (beta-CATENIN) gene itself. Recently in Wilms tumors, WTX (Wilms tumor gene on the X-chromosome) was discovered as another gene involved in the destruction of beta-CATENIN. As the WTX-gene harbors a short T(6)-microsatellite in its N-terminal coding region, we hypothesized that frameshift-mutations might occur in MSI-H CRCs in the WTX gene, thus additionally contributing to the stabilization of beta-CATENIN in human CRCs. Methods: DNA was extracted from 632 formalin-fixed, paraffin-embedded metastatic CRCs (UICCIV) and analyzed for MSI-H by investigating the stability of the highly sensitive microsatellite markers BAT25 and BAT26 applying fluorescence capillary electrophoresis (FCE). Then, in the MSI-H cases, well described mutational hot spot regions from the APC-, AXIN2- and CTNNBI genes were analyzed for genomic alterations by didesoxy-sequencing while the WTX T(6)-microsatellite was analyzed by fragment analysis. Additionally, the PCR products of T(5)-repeats were subcloned and mutations were validated using didesoxy-sequencing. Furthermore, the KRAS and the BRAF proto-oncogenes were analyzed for the most common activating mutations applying pyro-sequencing. mRNA expression of WTX from MSI-H and MSS cases and a panel of colorectal cancer cell lines was investigated using reverse transcription (RT-) PCR and FCE. Results: In our cohort of 632 metastatic CRCs (UICCIV) we identified 41 MSI-H cases (6.5%). Two of the 41 MSI-H cases (4.8%) displayed a frameshift mutation in the T(6)-repeat resulting in a T(5) sequence. Only one case, a male patient, expressed the mutated WTX gene while being wild type for all other investigated genes. Conclusion: Mutations in the WTX-gene might compromise the function of the beta-CATENIN destruction complex in only a small fraction of MSI-H CRCs thus contributing to the process of carcinogenesis

CXCL12 inhibits expression of the NMDA receptor's NR2B subunit through a histone deacetylase-dependent pathway contributing to neuronal survival

Homeostatic chemokines, such as CXCL12, can affect neuronal activity by the regulation of inhibitory and excitatory neurotransmission, but the mechanisms involved are still undefined. Our previous studies have shown that CXCL12 protects cortical neurons from excitotoxicity by promoting the function of the gene-repressor protein Rb, which is involved in the recruitment of chromatin modifiers (such as histone deacetylases (HDACs)) to gene promoters. In neurons, Rb controls activity-dependent genes essential to neuronal plasticity and survival, such as the N-methyl--aspartic acid (NMDA) receptor's subunit NR2B, the expression of which in the tetrameric ion channel largely affects calcium signaling by glutamate. In this study, we report that CXCL12 differentially modulates intracellular responses after stimulation of synaptic and extrasynaptic NMDA receptors, by a specific regulation of the NR2B gene that involves HDACs. Our results show that CXCL12 selectively inhibits NR2B expression in vitro and in vivo altering NMDA-induced calcium responses associated with neuronal death, while promoting prosurvival pathways that depend on stimulation of synaptic receptors. Along with previous studies, these findings underline the role of CXCL12/CXCR4 in the regulation of crucial components of glutamatergic transmission. These novel effects of CXCL12 may be involved in the physiological function of the chemokine in both developing and mature brains

Microsatellite instability in colorectal cancer is associated with local lymphocyte infiltration and low frequency of distant metastases

Colorectal carcinomas (CRCs) with high microsatellite instability (MSI-H) share clinicopathological features distinctly different from their microsatellite stable (MSS) counterparts. Unlike MSS cancers, MSI-H CRCs occur predominantly in the right-sided colon and are often characterised by a strong lymphocyte infiltration. A poor differentiation pattern is found in most MSI-H CRCs, even though patients with MSI-H carcinomas seem to have a significantly longer survival after surgical resection. To clarify which factors contribute to the obvious paradoxon of a more favourable prognosis of MSI tumours, several clinical and histopathological features as well as the microsatellite status were evaluated in 120 colorectal cancer cases fulfilling clinical criteria (Bethesda) indicative for familial colorectal cancer. Microsatellite instablity status and lymphocyte infiltration were related to tumour stage and patients' follow-up. Statistical analysis confirmed well-known relations, such as enhanced lymphocyte infiltration accompanied by Crohn's like reaction (CLR) in MSI-H cancers (CLR+ in 27 out of 47 MSI-H vs 14 out of 71 MSS CRCs, P<0.001). However, after stratification for depth of local invasion and penetration of the primary tumour, T3 tumours displaying MSI had a significantly lower rate of distant metastases (M1 in four out of 35 MSI-H vs 20 out of 41 MSS CRCs, P<0.001). A similar tendency was observed for CLR-positive CRCs (M1 in six out of 29 CLR+ vs 17 out of 45 CLR− CRCs, P=0.13). In a logistic regression model, the MSI-H phenotype and the presence of CLR were independent predictors of a low UICC stage (P=0.006 and 0.04, respectively). These data, together with the recent definition of highly immunogenic neo-antigens expressed in MSI-H tumour cells, suggest that MSI-H CRCs elicit a protective host response that may prevent metastasis formation

Cancer Genes Hypermethylated in Human Embryonic Stem Cells

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation